Filtering strategies are a crucial aspect for signal detection in many fluorescence based systems such as chemical and/or biochemical sensors. The design, fabrication and characterization of a new waveguide absorption filter for the optimization of the fluorescence signal collection, thanks to its high numerical aperture, is here presented. The absorption filter is designed to work as an optical waveguide in order to increase the optical path and, consequently, the absorption of the excitation light. A comparison of the performances of the absorption filter and a conventional interference filter, with particular emphasis on the angular dependence of the spectral features, is also reported. We experimentally demonstrate, for what regards the attenuation capability of the excitation signal, the failure of the interference filter for incidence angles greater than 15° and the validity of the absorbing waveguide filter for large incidence angles. Finally, preliminary results performed in fluorescence on an IgG labelled/ anti-IgG assay show the improvement in detected fluorescence intensity collected by means of the proposed absorption filter compared to that measured with the interference filter. This suggests that the filtering strategy based on the waveguide absorption filter can greatly simplify fluorescence detection systems and find interesting applications in different areas of sensing, from Point of Care Testing (POCT) to environmental monitoring.
Infectious diseases and sepsis, as a severe and potential medical condition in which the immune system overreacts and finally turns against itself, are a worldwide problem. As a matter of fact, it is considered the main cause of mortality in intensive care. For such a pathology, a timely diagnosis is essential, since it has been shown that each hour of delay in the administration of an effective pharmacological treatment increases the mortality rate of 7%. Therefore, the advent of a POCT platform for sepsis is highly requested by physicians. Biomarkers have gained importance for the diagnosis and treatment monitoring of septic patients, since biomarkers can indicate the severity of sepsis and can differentiate bacterial from viral and fungal infection, and systemic sepsis from local infection.
The present paper deals with the development of fluorescence-based bioassays for the sepsis biomarkers and their integration on a multianalyte chip. Among the different biomarker candidates, the attention was focused on procalcitonin (PCT), C-reactive protein (CRP) and interleukine-6 (IL-6) as well as on soluble urokinase plasminogen activator receptor (suPAR) recently proposed as a very effective inflammatory marker, potentially capable of acting also as a prognostic biomarker. Starting point of this new setup was an already developed fluorescence-based optical platform, which makes use of multichannel polymethylmetacrylate chips for the detection of different bioanalytes, and the serial interrogation of the microfluidic channels of the chip. The novel proposed optical setup makes use of a suitable fluorescence excitation and detection scheme, capable of performing the simultaneous interrogation of all the channels. For the excitation part of the optical setup, a diffractive optical element is used which generates a pattern of parallel lines, for the simultaneous excitation of all the channels and for the optimization of the optical power distribution. For the detection part, an array of optical absorbing waveguides (long-pass coloured glass filters) is used, which collects the scattered light and the emitted fluorescence, filters out the excitation component, and is faced to a large area rectangular detector, for the simultaneous fluorescence detection. The implemented sandwich immunoassays comprise a capture antibody immobilized onto the surface of the chip channel and a detection antibody properly labelled with a fluorophore. Limits of detection of 2.7 ng/mL, 0.022 µg/mL, 12 ng/mL and 0,3 ng/mL were achieved for PCT CRP, IL-6 and suPAR, respectively.
The realization of a simple real time biosensor, in which antibodies are immobilized onto surfaces, represents a promising application in the immunoassay development. Among the various sensing approaches, one of the most promising is based on microring resonators, offering a lot of advantages such as mass production, reduced dimensions, label-free and real time detection. The use of the evanescent field as optical transduction principle allows the development of label-free biosensors, in which the antibody is usually immobilized on the sensor surface and the binding of the antigen can be controlled and followed in real-time.
The overall performances of immunosensors are strongly related to the optimization of the immobilization process and the integration between the microfluidic parts and the optical detection system. The combination of these two aspects makes the biosensing process very efficient, with a consequent reduction of the response time and improvement of the immobilization process efficiency.
In this work we explore the working mechanism of a flow-through microresonator platform. A drilled hole, in the center of the ring, allows the active transport mechanism of the analyte toward the sensing surface with a consequent reduction of the response time. Moreover, we study the effects of oxygen plasma, in terms of duration times and plasma power, on immobilization efficiency of immunoglobulin G (IgG). An improvement of about 20% of the protein adsorption is ascribed to chemico-physical modification of SU-8. The measured sensor response time in flow-through configuration is about five times shorter respect to standard flow-over configuration.
A novel therapeutic drug monitoring point of care testing (POCT) optical device for the detection of immunosuppressants in transplanted patients was designed and tested, with the body interface constituted by an intravascular microdialysis catheter (MicroEye®) which provides the dialysate as clinical sample. An optical biochip with 10 microchannels, based on total internal reflection fluorescence (TIRF), enables the frequent measurement of immunosuppressants. Heterogeneous competitive immunoassays for the detection of mycophenolic acid, tacrolimus and cyclosporine A are implemented on the different microchannels, with the derivative of the immunosuppressants immobilised on the bottom part of the micro-channels.
In recent years, microbottle resonators that support non-degenerate whispering gallery modes (WGMs), propagating by successive total internal reflections close to the resonator surface and all along its axis, have been widely investigated due to their potential applications in optical sensing, microlasers and nonlinear optics. To overcome some drawbacks of the standard silica microbottle resonators, we focused our attention on polymers such as SU-8 resist and NOA resins. A drop of polymeric material is dispensed onto a fiber stem, providing a mechanical support for the bottle resonator, and is photo-polymerized by an UV lamp. The interrogation system, usually constituted by a tapered silica fiber evanescently coupled with the microresonator, is substituted by a more stable planar waveguide realized in SU-8 by means of standard photolithography technique. Moreover, for guarantying the stability to surrounding disturbance of the coupling between the microbottle resonator and the planar waveguide, the fiber stem is glued to substrate. Two drilled holes in the substrate allow the rise of the glue at the ends of the fiber stem and the fixing of sensor on PMMA substrate.
In the present work, we presented an integrated full polymeric platform with self-assembled bottle microresonators packaged in a stable structure. SU-8 and NOA based microbottles are realized and morphologically characterized. The low autofluorescence emission and long term stability make the NOA based bottles suitable to be employed in a great variety of conditions. Bulk sensing measurements are performed by using water:ethanol solutions and a bulk sensitivity of 120 nm/RIU is estimated.
Polymer micro-bottle resonators are fabricated by means of a simple self-assembling process. High optical quality SU-8 negative resist is chosen as bottle material. SU-8 is dispensed onto a fiber stem and is kept in rotation during the UV photo-polymerization. Micro-bottles with different geometrical sizes have been obtained by changing the SU-8 dispensed volume, with a repeatability of about 2%. Planar waveguides is chosen as interrogation system for our SU-8 self-assembled micro-bottle resonators. The highest quality factor Q associated to the resonance peak in the transmission spectrum is about 3.8 × 104.
A novel fuel level sensor for aeronautical applications is developed. The sensor is based on an array of total internal reflection (TIR) point sensors. Respect to conventional TIR sensors the new design permits to be sensitive to common jet fuels (JetA, JP4,JP7) but also to operate with new alternative fuels. The sensor doesn’t require aircraft calibration, temperature compensation and furthermore is able to operate correctly when partially or totally exposed to presence of condensed water on its surface. The point sensors are multiplexed on a single fiber by optical couplers and interrogated simultaneously by Optical Time Domain Reflectometry (OTDR) at a wavelength of 1550nm. Experimental results show a resolution of ±1.5mm could be achieved. The sensors is also able to measure the free water level in the fuel.
Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory protein present in blood and a marker of
disease presence, severity and prognosis. A heterogeneous sandwich assay is proposed for quantifying suPAR by
employing a capture antibody from rat and a biotinylated detection antibody from mouse. Optical detection was achieved
by a successive exposure of the biotinylated sandwich to streptavidin labelled with ATTO647N. The heterogeneous
assay was implemented on a multichannel polymethylmetacrylate (PMMA) optical biochip, potentially capable of the
simultaneous detection of more than one analyte. Capture antibody was immobilized on the PMMA surface of the
microfluidic channel and the assay was performed with the following protocol: i) surface blocking with BSA, ii)
incubation with suPAR or PBS, iii) incubation with biotinylated suPAR detection Ab and iv) incubation with
streptavidin-ATTO647N. Promising preliminary results were obtained with this protocol. Moreover, an improved optical
setup is proposed which avoids the mechanical scanning of the chip and consequently the in-series fluorescence
excitation and read out, allowing the simultaneous measurement of the fluorescence on all the channels of the
In this work we show that integrated Hybrid Silicon-PDMS Antiresonant Reflecting Optical Waveguide (H-ARROW) can be applied for the realization of optofluidic devices. H-ARROW is constituted by the optofluidic channel of a conventional ARROW, sealed with a thin PDMS layer. This layout simplifies the integration of microfluidic parts to manipulate liquid samples, which can be easily fabricated in the PDMS layer. Hybrid ARROWs have been fabricated and used in order to design complex devices like an integrated hybrid liquid core optofluidic ring resonator (h-LCORR) and a hybrid optofluidic platform for fluorescence measurements.
An optofluidic hybrid platform based on hybrid liquid core ARROW waveguides has been fabricated and tested. Solid core hybrid ARROW was integrated in a self-aligned optical configuration with the ARROW optofluidic channel for an improved collection efficiency. The platform was fabricated using a modular approaches. The microfluidic system was completely realized with PDMS using a layered structure while the optical part was realized developing a hybrid silicon/PDMS solution. The performance of the system has been tested by carrying out fluorescence measurements on Cy5 water solutions, obtaining an LOD of 2.5 nM.
An optofluidic jet waveguide for Raman spectroscopy is reported. In this device a micro-channel is used to produce a
high speed liquid stream acting at the same time, as the solution to analyse and as an optical waveguide. The liquid
waveguide, exploiting total internal reflection, is able to effectively collect the Raman signal produced by the chemical
compound under analysis opportunely excited by means of a laser source. Using a self-aligned configuration, the liquid
jet is directly coupled with a multimode optical fiber collecting the Raman signal towards the detection system. The
waveguiding nature of a liquid jet enables high Raman signal collection and the device configuration allows strong
reduction of the background as no confining walls are used to contain the solution to analyse. The performances of the
system have been successfully tested on isopropyl alcohol in water solutions showing a detection limit for this chemical
compound of 0.8±0.1%.
The design of a novel therapeutic drug monitoring (TDM) point-of-care-testing (POCT) biochip for immunosuppressants
detection in transplanted patients is described. The chip consists of two polymeric parts, a top PMMA slide and a bottom
ZEONOR® thin foil, bonded together by means of a pressure sensitive adhesive tape. The tape, with lower refractive
index, is shaped in order to obtain a microfluidic multi-channel array. The optical signal, coming from an external light
source and travelling along the ZEONOR® thin foil, excites the fluorescent sensing layer immobilized onto the fluidic
channels. Preliminary tests with the bioassay implementation for tacrolimus detection are reported.
Over the last years optofluidics has emerged as a very promising field. The integration at microscopic level between
optics and microfluidics provides a number of unique characteristics that cannot be obtained with solid materials only.
This paper briefly reviews the state of the art on optofluidics for sensing applications. We first describe the different
approaches in order to realized optofluidic waveguides. These waveguides allow an increased interaction efficiency
between the light and the liquid substance that can be very useful in sensing applications (fluorescence, absorption
spectroscopy, etc.). We then illustrate high sensitive optofluidic devices such as Mach-Zehnder interferometers, Fabry-
Perot cavities and ring resonators. Examples of applications of optofluidic sensors for chemical and biological analysis
An optofluidic jet waveguide for on chip fluorescence analysis is presented. The waveguide consists of an high speed water jet produced by means of a micro-channel coupled with a multimode optical fiber collecting the fluorescence opportunely excited. The liquid jet acts, at the same time, as the solution to analyse and as an optical waveguide. This configuration allows a strong reduction of the scattering and fluorescence of non analyte substances enabling a very low limit of detection (LOD). The integrated device is fabricated by PMMA micro-machining allowing a self-alignment between the liquid jet waveguide and the optical fiber used to deliver the fluorescence to the detector. The performance of the system has been tested on Cy5 water solutions and LOD of 2.56 nM has been obtained. A proof-of-concept of filter-free measurements has been performed demonstrating that fluorescence measurements can be performed also by using a photodiode with an LOD of 6.11 nM.
In this work an integrated tunable optofluidic liquid core-liquid cladding (L2) optical fiber is presented. The device has been realized by exploiting an innovative three-dimensional hydrodynamic focusing scheme. A tunable circular liquid core has been obtained that is located in the center of the channel, regardless of the flow rate ratio of the cladding and core liquids. This circular geometry allows a more simple control of the optical property of the beam and the input and output coupling with standard optical fiber. A liquid core with a tunable diameter ranging from 45.3 to 11.2 μm has been successfully obtained.
In this work we review recent results of our work on optofluidics. We show that single mode optofluidic waveguides
with low loss can be realized using antiresonant reflecting optical confinement (ARROW). These waveguides can be
fabricated using full standard silicon technology or hybrid silicon-polymer processes. ARROW waveguides have been
used in order to realize complex optofluidic devices like Mach-Zehnder interferometers and ring resonators. The
advantage of using optofluidic waveguide results in very compact devices with a total length of 2.5 mm and a required
liquid volume less then 0.16nl.
This paper reports a micro flow cytometer fabricated in Polymethylmethacrylate (PMMA) in which a 3d hydrodynamic
flow focusing is employed in order to align the particles in a single line along the focused stream. The device has been
fabricated by direct micro milling of two parts of PMMA that were finally bonded together. With a suitable choice of the
fluidic channel geometry, a circular sample stream located in the center of the channel is obtained. Numerical
simulations have been performed in order to investigate the flow characteristic of the structure and find the desiderated
geometry. Three dimensional hydrofocusing of the sample fluid was analysed and demonstrated by cross sectional
fluorescence imaging in good agreement with numerical simulations. Flow cytometry measurements have been
performed by using 10μm particles. From the analysis of the fluorescence signals collected at each transit event we can
confirm that the device was capable of creating a single-file particle stream. The results show that the device was capable
of discriminating single microparticles with a good signal-to-noise ratio and a high throughput.
We report the design and the fabrication of an integrated optofluidic ring resonator based on liquid core antiresonant
reflecting optical waveguide (ARROW) operating around λ=635nm with a water filled core (nc=1.33). This waveguide
permits a strong coupling between light and analyte as the same microchannel can be used for delivering sample and for
confining light. A rectangular-shaped ring resonator has been realized by using four 90-bent waveguides and an
multimode interference coupler is used for coupling light from the input bus waveguide to the resonator. The resulting
device is very compact, with a total length of 2.5 mm and a required liquid volume of about 0.11nl. The device has been
simulated with a two dimensional finite-difference time-domain method for different refractive index core nc=1.32
(methanol) and nc=1.43 (dimethylformamide). Moreover, we carried out optical measurements by measuring the
transmitted spectrum from the output bus waveguide in both cases. The experimental results confirm that ring with a
good quality factor (Q~800) can be achieved in good agreement with the numerical results.
Integrated liquid core AntiResonant Reflecting Optical Waveguide (ARROW) are used as basic component for the
realization of complex optofluidic devices. Liquid core ARROW waveguides permit to confine the light in a low
refractive index liquid core, by means of two high refractive index cladding layers designed to form a high reflectivity
Fabry-Perot antiresonant cavity. This arrangement allows to realize liquid core waveguides that can be very useful in
optofluidic applications. We report the fabrication and the characterization different optofluidic devices based on hollow
core ARROW waveguide like tuneable couplers and Mach-Zehnder interferometers. The proposed devices have been
realized by silicon technology. The channels have been realized by etching the silicon wafer, while the two claddings
have been deposited on both wafers by LPCVD or ALD depositions.
We propose the use of perfluorinated cladded multimode polymer optical fibers tapers for fluorescent detection in
aqueous environment. A graded-index polymer optical fiber with core and cladding diameters of 62.5/90 and very low
refractive index (nCore = 1.356, nCladding = 1.342) has been used. The taper has been fabricated using the heat-and-
pull technique. Despite to the fact that taper core is not in direct contact with the external medium, this fiber taper can be
used for sensing applications. In fact, some of the guided modes are no more confined in the core region but can still be
guided by the fiber in the cladding region. Therefore, in the taper region, there is an evanescent wave in the external
medium, related to the cladding modes. The taper geometry and the very low refractive index of the material corecladding
(1,35-1,34) permits a strong enhancement of the penetration depth increasing the fluorescence collection
efficiency in aqueous environments (1.33). Fluorescence measurements in an aqueous solution containing Cy5 dye in a
concentration range 3.14×10-7M to 6.76×10-6M have been performed.